We have recently showed that 11-cis retinal, the light-detecting chromophore of the visual pigment, is a major source of the toxic deposits that originate in the photoreceptors and accumulate in the Retinal Pigment Epithelium in the form of lipofuscin. This work was carried out with mice, and we now propose to extend these studies to the human. Our finding indicates that 11-cis retinal may play a major role in events resulting in retinal degenerations. Defects of retinoid processing in the eye have long been linked to diseases of the retina, but most pathogenic mechanisms have been hitherto associated with either lack of 11-cis retinal or accumulation of its photoproduct all-trans retinal. Here we propos to examine the damage to the human retina initiated by 11-cis retinal. This is particularly important for the human retina, where rhodopsin regeneration is rapid, requiring high fluxes of 11-cis retinal. We will investigate the damage mediated by 11-cis retinal in single living rod photoreceptors from human donor eyes. Mouse rods will be used for comparison, in order to aid in the extrapolation of results from whole animal studies with mice to the human situation. We will use fluorescence imaging of single photoreceptors to measure oxidative damage and the formation of lipofuscin precursors. To determine the relation between the flux of 11-cis retinal and the rate of rhodopsin regeneration, we will measure rhodopsin levels with microspectrophotometry. The aims of the research are: Specific Aim #1: Test if sequestration of 11-cis retinal prevents damage to the rod outer segment. Specific Aim #2: Test if increases in the 11-cis retinal flux increase damage to the rod outer segment. Results from these studies will provide new insights into the basic pathogenic mechanisms underlying vision loss in diseases like Age-Related Macular Degeneration and Stargardt, and suggest possible pathogenic mechanisms for a host of other diseases that might involve the generation and delivery of 11-cis retinal. They will elucidate the process of rhodopsin regeneration, which is essential for the recovery of visual sensitivity after light exposure. By using living human rod photoreceptors, the experimental approach is uniquely able to obtain mechanistic information about the origins of lipofuscin and the process of rhodopsin regeneration in the human eye. These studies will allow the evaluation of the potential toxicity of therapies for 11-cis retinal deficiencies that depend on boosting the chromophore supply. They will also provide a measure for the effectiveness of the opposite type of therapies, which aim to limit lipofuscin formation by slowing down the generation of 11-cis retinal.